An ultrafiltration assay for nucleotide binding to ribonucleotide reductase.
Direct partition through ultrafiltration was applied to develop a method for the study of nucleotide binding to ribonucleotide reductase from Escherichia coli. The assay involved a 0.5- to 1-min centrifugation step where bound and unbound nucleotides are separated over an ultrafiltration membrane. No effects were seen due to hyperconcentration of protein at the membrane surface. The method was verified by measuring binding of dATP, ATP, dTTP, dGTP, and GDP at 25 and 4 degrees C with dissociation constants ranging from 0.1 to 80 microM. The results were in good agreement with earlier data obtained by other techniques and extend our knowledge in the case of ATP and dGTP binding at 25 degrees C.
Ormö
M
M
Department of Molecular Biology, University of Stockholm, Sweden.
Sjöberg
B M
BM
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis; D002498:Centrifugation; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000378:metabolism; D014462:Ultrafiltration / Q000379:methods
D001426:Bacterial Proteins; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D009711:Nucleotides; D011506:Proteins; D013942:Thymine Nucleotides; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases; C024157:thymidine 5'-triphosphate